DNA gyrase of Mycobacterium tuberculosis will be studied to better understand acquisition of antibiotic resistance and factors that determine intracellular DNA structure in mycobacteria. The two gyrase genes of M. tuberculosis will be cloned, and their nucleotide sequences will be determined to provide molecular information needed for analysis of fluoroquinolone resistance. Then the gyrase genes from fluoroquinolone- resistant clinical strains will be examined for mutations that will account for resistance. Gyrase genes cloned from these strains will be used to express gyrase, which will then be purified and studied in vitro for resistance properties. To determine whether gyrase is required for resistance, genetic complementation will be carried out using cloned, wild-type gyrase genes introduced into quinolone-resistant clinical strains. Inhibition of DNA synthesis in permeabilized cells will be examined to identify strains in which resistance is due to non-gyrase determinants. Gyrase inhibitors will also be used to obtain additional mutants that have altered steady-state levels of supercoiling. These will be used to ask whether perturbation of supercoiling interferes with virulence by examining the ability of mutants to inhibit mycobacterial survival and reproduction in cultured macrophages. Information obtained from this program will define quinolone resistance in M. tuberculosis and will begin to describe relationships between DNA supercoiling and virulence in M. tuberculosis. The work is expected to help guide efforts to develop new, more effective anti-tuberculosis agents.